【作者】 孙德光；

【导师】 王立明；

【作者基本信息】 大连医科大学 ， 外科学， 2010， 博士

【摘要】 肝细胞肝癌(hepatocellular carcinoma, HCC)是全球恶性程度极高、预后极差的恶性肿瘤之一。我国由于肝炎和肝炎转肝硬化的病人众多致使肝癌在我国的发病率和病死率均高于世界平均水平,全世界50%以上的肝癌发生在我国,每年约有23万人死于肝癌,位列我国癌症病死率的第二位,占我国全部恶性肿瘤死亡人数的18.8%。目前肝癌发生发展及转移复发的精确分子机制尚不完全清楚。临床主要依靠定期复查AFP和B超来检测肝癌和术后转移复发情况,但在中国AFP阳性的肝癌只有70-80%,而B超难以发现小于l cm的肝癌组织,诊断均存在盲区,不能准确及时地进行预测。因此探讨肝癌发生发展及转移复发的相关因素及分子机理,揭示其发生发展及转移复发的精确分子机制、寻找早期诊断肝癌、预测转移的生物标记物和干预治疗的靶分子,对进一步提高我国肝癌的治疗水平具有重要意义。蛋白质组已经成为系统生物学时代最热点的研究领域之一。蛋白质组学技术也随之不断发展完善,并与各个学科交叉而产生新技术、新方法。这些蛋白质组学的新技术正逐渐成为现代生物医学研究新的强有力工具。肿瘤的基础研究随之也进入了一个全新的领域。但蛋白的功能仅仅从量的变化上分析往往会限制我们的研究视野,很多重要蛋白质的活性是由翻译后修饰调节的,甚至有时蛋白水平上看不到明显变化但翻译后修饰水平已发生显著变化。蛋白质的磷酸化修饰是生物体内最重要的共价修饰方式之一,参与调节细胞的多种生命活动过程,包括细胞的增殖、发育和分化、细胞骨架调控、细胞凋亡、神经活动、肌肉收缩、新陈代谢、肿瘤发生等。鉴于蛋白质磷酸化修饰在生命活动中所具有的重要意义,解密蛋白质磷酸化修饰过程的奥秘以及进一步理解蛋白质磷酸化介导的生物学功能成为了众多生物学家所关心的主题。并且研究不同生理条件下的生物体蛋白质磷酸化修饰的状态,能够更清晰地认识生物体信号通路的变化,继而揭示疾病产生的机理。但由于生物体内磷酸化蛋白质的含量及磷酸化位点的化学计量值常常很低,大规模分析磷酸化蛋白质在技术上还很具有挑战性。所以磷酸化蛋白质组学的研究发展缓慢,与肝癌相关磷酸化修饰谱的规模化分析鉴定在国内外更是鲜为报道。课题组在前期工作中研发出许多磷酸化蛋白质检测的创新技术,将这些磷酸化蛋白质检测技术用于临床疾病——肝细胞肝癌研究中,有利于揭示肝癌的发病机制,寻找肝癌诊断和治疗的靶点。目的：1.首先全面、系统的考察Ti4+-[MAC对磷酸肽富集的选择性和特异性。2.利用Ti4+-IMAC富集实际样本人肝癌组织及正常肝组织酶解后的磷酸肽,结合LC-MS2-MS3检测和数据自动检索等一系列蛋白磷酸化检测技术规模化筛选人肝癌组织和正常肝组织中磷酸化蛋白质表达谱,寻找可能参与肝癌发生发展的磷酸化蛋白质。3.探讨人肝细胞肝癌组织中差异磷酸化蛋白(PED／PEA-15.XIAP.P27-T 187)的表达情况及其与肝细胞肝癌临床病理特征的关系。方法：1.以标准磷酸化蛋白质β-酪蛋白(β-casein)和α-酪蛋白(α-casein)为样品,利用MALDI-TOF MS质谱对比分析Ti4+-IMAC富集磷酸肽效果。2.收集12例肝细胞肝癌和10例人正常肝组织,提取总蛋白,经胰蛋白酶酶解后,利用Ti4+-IMAC富集实际样本人肝癌组织及正常肝组织酶解后的磷酸肽,结合LC-MS2-MS3检测和数据自动检索等一系列蛋白磷酸化检测技术规模化筛选人肝癌组织和正常肝组织中磷酸化蛋白质谱,结合生物信息方法预测蛋白的磷酸化位点及蛋白激酶等信息,寻找可能参与肝癌发生发展的差异磷酸化蛋白。3.挑选其中三种差异磷酸化蛋白,通过免疫组织化学、Western blot及RT-PCR分子生物学方法,观察其在40例癌组织及对应癌旁组织和12例正常肝组织中的表达差异。结果：1.β-酪蛋和α-酪蛋白酶解液经过Ti4+-IMAC单分散微球富集后MALDI-TOF质谱图中均检测到很强的磷酸化肽段质谱峰；Ti4+-IMAC单分散微球在模拟复杂体系中也表现出良好的特异性；在模拟复杂样品加中磷酸化酪氨酸肽段,经Ti4+-IMAC单分散微球富集后,MALDI-TOF质谱图依然检测到了pY质谱峰。2.利用Ti4+-IMAC富集肝癌组织中磷酸化多肽,经LC-MS2-MS3鉴定、数据库检索和生物信息学分析,在12例肝癌组织中,高信度得鉴定到了240个磷酸化肽段,共169个磷酸化位点,149个磷酸化蛋白,其中包括细胞骨架蛋白、信号传导蛋白、酶解相关蛋白及一些功能未知蛋白等。对比分析正常肝组织中磷酸化蛋白表达,成功鉴定到了包括细胞骨架类、细胞周期、凋亡类及亚细胞器类等20个差异磷酸化蛋白。结合SCANSITE(http://scansite.mit.edu)分析软件预测,人肝癌组织中的磷酸化蛋白主要是由蛋白激酶CK2磷酸化。3.选取三种差异磷酸化蛋白,进一步利用分子生物学方法,验证其在肝组织、癌旁组织及正常肝组织中的表达,免疫组化及Western blot结果提示PED／PEA-15(s-116)、XIAP(s-87)和P27-T187在肝细胞肝癌组织表达均明显高于癌旁、正常肝组织,有高度显著性差异(P<0.05),三者均与肝癌病理分级、临床分期相关,在肿瘤分化程度越差、临床晚期肝癌组织表达增加,而且强度增强(P均<0.05)。而与患者的发病年龄、性别、肿瘤大小、肿瘤数目及复发转移均无显著差异性(P均>0.05)。PED／PEA-15(s-116)和XIAP(s一87)蛋白的表达为正相关,结果为r=0.371,P=0.018。结论：1.Ti4+-IMAC单分散微球对磷酸化肽段的富集有非常高的特异性和选择性,是用于磷酸化蛋白质组学研究理想的IMAC材料。2.Ti4+-IMAC富集,LC-MS2-MS3分析,结合数据库自动检索和生物信息学分析这一新的磷酸化蛋白质组学检测技术路线,为大规模磷酸化蛋白质组的分析提供了一个强有力的手段,可能成为探索肿瘤发病机制提供有力的工具。3.在肝癌组织中鉴定到的149个磷酸化蛋白以及对应磷酸化位点和蛋白激酶等的这一数据库信息,以及筛选的20个差异磷酸化蛋白,可能为探索肝癌发病机制和寻找新的治疗靶点提供有价值的信息。4.利用免疫组织化学及Western blot的方法对包括PED／PEA-15(s-116)、XIAP(s-87).P27-T187在内的三种磷酸化蛋白进行了验证,蛋白变化趋势与质谱鉴定结果一致。PED／PEA-15(s-116).XIAP(s-87).P27-T187蛋白与基因mRNA在肝细胞肝癌中高表达,可能与肝癌的发生发展密切相关。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignant tumors.for more patients with hepatitis and cirrhosis,themotbidity and mortatity of HCC are higher than that of the world average in china,more than 50%HCC worldwide occur in china,which causing 23 million people’s death each vear. HCC is the second-leading cause of cancer death and accounting for 18.8% in the death of malignant tumors in china. Currently, the underlying molecular mechanisms of hepatocarcinogenesis that lead to malignant transformation of normal liver cells and metastasis of tumor cells remain unclear. The detection and anticipation of HCC and postoperative recurrence and metastasis majorly depend on AFP and type-B ultrasonic is unavailable to the tumor smaller than 1 cm. So there is a blind are a in diagnosis and it is hard to make an accurate anticipation. Therefor that studing and revealling the exact molecule mechanism of occurrence and development of HCC, exploring novel tumor markers and targets of drug therapy for tumor is important for the cure of HCC.Proteomic becomes one of the most popular researching area of systemic biology. With the development of proteomic and its crossover with other subjects, new technology and researching ways has been found. Those technology gradually play as an important role in modern bio-medical research. Basic researches of tumor steps into an brand new world. It is usually limited our vision by study protein function merely on the quantity change level. Most protein’s activities infected by modification and regulation after translation. Sometimes protein modification dramatically changed without any quantity change. The modification of protein phosporylation is the most important way of covalence modification in human body, which participate in most life activities. Those activities include cell proliferation, development and differentiation; cellular structure regulation; cell atrophy; nerve activities; muscle contraction; metabolism; tumor genesis etc. considering about the important meaning of phosphorylation modification, more and more biological scientists star to work hard in order to explore the unknown process of protein phosphorylation and the biological function that mediated by the protein phosphorylation. Understanding the different state of protein phosphorylat-Ion in different physiological situation can make biological signal pathway alternation clear. Furthermore, It can lead us to discover the mechanism of disease genesis. However, the concentration of phosphoryprotein is pretty low and the quantity of phosphoryprotein’s chemical site is very little,which remains many challenges for the large scale analyze of phosphoryprotein. So proteomics of phosphoryprotein developing slowly, and its association with hepatic cancer has not been reported all over the world. Now we found some new researching technology during the pre-researching period, Those technology can be used in examine clinical disease, such as hepatocellular carcinoma. It brings a lot of benefits in discovering the mechanism if hepatic cancer genesis and finding the target for cancer diagnosis and treatment.Purpose:1. full-scale and systemic study the specificity and the alternation of phosphopeptide’s enrichment by Ti4+-IMAC.2.analyzing the phosphory-Protein expressing profile of both normal liver tissue and hepatic cancer tissue by using the method Ti4+-IMAC combining with LC-MS2-MS3 and database research. Large-scale and high-flux screening of differential phosphoprotein, identifying the phosphorylated sites and protein kinase by using biological information.3. investigate the expression of differential phosphoprotein(PED/PEA-15, XIAP, P27-T187) in tissue of hepatocellular carcinoma and explore its relationship to clinicopathological features of HCC.Methods:1. the standard phosphoprotein(a-casein andβ-casein) wss used as sample,compare and analyze the enrichment effection of Ti4+-IMAC by MALDL-TOF MS.2. analyze the phosphoprotein expressing profile, phosphorylated sites and prorein kinase in both hepatic cancer tissues(12 samples) and normal liver tissue(10 samples) by the method Ti4+-IMAC combining with LC-MS2-MS3 and datebase research,in order to find differential phosphprotein.3. pick up 3 kinds of differential phosphprotein to study the differences between 40 hepatic cancer tissue as well as its nearby hepatic tissue and another 12 normal liver tissue by using IHC,Western blot and RT-PCR.Result:1.the enrichment of phosphopeptide is available and specificity by Ti4+-IMAC.2.240 phosphopeptides,169 phosphorylated sites and 149 phosphprotein including cytoskeletal protein, sigal conduction protein, enzymolysis protein and other unknown proteins were found by the method Ti4+-IMAC combining with LC-MS2-MS3 and datebase research. Further more,20 differential phosphproteins were indentified including cystoskelet-on, cell cycle, Apoptosis and deuto-cell from hepatic cancer comparing with the normal liver tissues.also it is found that the phosphproteins in hepatic cancer were major actived by protein kinase CK2.3. the 3 protein(PED/PEA-15(s-116)、XIAP(s-87), P27-T187) were high expressed in Hepatic cancer comparing with nearby hepatic tissue and normal liver tissues by using IHC and Western blot(P<0.05). And The expression of PED/PEA-15(s-116) was significantly associated with pathological grade, clinical stage in HCC(P<0.05), however it was not significantly correlated to other clinicop-athological features(P>0.05). the expression of PED/PEA-15(s-116) and XIAP(s-87) was positive correlation, (r=0.371, P=0.018).Conclution:1. Ti+-IMAC is available to enrichment phosphopeptides for its high specificity and alternative, which can be an ideal material for the study of ph osphoproteomic.2. the method Ti4+-IMAC combining with LC-MS2-MS3, database research and Bioinformatics provides a drastic perspective in Phosphoproteomic, which may be an important tool to explore the pathogenesy of tumor.3. the phosphprotein datebase (149 phosphproteins, phosphorylated sites and protein kinase) and 20 differential phosphproteins may paly an important role in exploring the hepatic cancer pathogenesy and providing a new drug therapy way.4. the expression of PED/PEA-15 (s-116)、XIAP(s-87)、P27-T187 is the same as MS by IHC and Western Blot,suggesting the high expression of those proteins may be closely realted to the occurrence and development of HCC.更多还原